Diphtheria, which re-emerged as a major epidemic disease in Russia andthe Newly Independent States of the former Soviet Union during the 1990s, is a paradigm for toxin-mediated bacterial disease. Tuberculosis, which causes more deaths worldwide than any other bacterial infection, is a paradigm for intracellular bacterial infection. Very similar iron-activated regulatory proteins, called the diphtheria toxin repressor (DbcR) and the iron-dependent regulator (IdeR), control virulence in C. diphtheriae and M. tuberculosis, respectively. Db<R and IdeR are prototypes for a family of metal-dependent global regulators predominantly found in Gram-positive and acid-fast bacteria. During the current project period, we used genetic and biochemcial methods to identify iron-regulated genes and gene products in C. diphtheriae, and we developed lead compounds that promote activation of DbcR or IdeR at lower concentrations of ferrous ions than are needed to activate DtxR or IdeR by itself. During the next project period we will extend these studies in several important ways. In Aim 1, using C. diphtheriae, we will characterize the structure and function of several important iron-regulated genes, determine the molecular basis for DtxR-dependent and Db<R-independent regulation of gene expression by iron, investigate the roles of specific sigma factors within the iron regulon, and investigate oxidative stress pathways and their interactions with iron-dependent gene regulation. In Aim 2, we will characterize the multiple siderophore-dependent iron-uptake pathways in C. diphtheriae that participate in maintenance of iron homeostasis. We will characterize interaction of diphtheriabactin (the siderophore produced by C. diphtheriae) with its dip0582/ciuA receptor, and we will identify the genetic systems that enable C. diphtheriae to use other siderophores (e.g., desferrioxamine, ferrichrome, rhizoferrin and the recently discovered siderophores from C. pseudotuberculosis). In Aim 3, we will apply structure- based methods to develop second-generation peptides and other molecules with high potency for stimulating or inhibiting Db<R or IdeR activity. We will test these novel compounds for biological activity, first in pound coli or C. glutamicum systems in which recombinant IdeR controls expression of reporter genes, and then in M. tuberculosis. Because ideR is essential in M. tuberculosis, and because expression of a hyper- repressor variant of DtxR attenuates the virulence of M. tuberculosis, we will determine whether the novel activators and inhibitors of IdeR that we are developing have antimicrobial activity against M. tuberculosis and potential value as chemotherapeutic agents for treatment of tuberculosis.